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The nitrogen (N) loss processes have not been well examined in subterranean estuaries (STEs) between land and sea. We utilized a 15N isotope tracer method, q-PCR, and high-throughput sequencing to reveal the activities, abundances, and community compositions of N loss communities in a STE in Gloucester Point, Virginia, US. The highest activities, abundances and diversity of denitrifiers and anammox bacteria were detected at 50–60 cm depth in the aerobic-anaerobic transition zone (AATZ) characterized by sharp redox gradients. nirS-denitrifiers and anammox bacteria were affiliated to 10 different clusters and three genera, respectively. Denitrification and anammox played equal roles with an estimated N loss of 13.15 mmol N m−3 day−1. A positive correlation between ammonia oxidizing prokaryote abundances and DO as well as NOx− suggested that nitrification produces NOx− which supports the hotspot of denitrification and anammox within the AATZ. Overall, these results highlight the roles of N loss communities in STEs